A neutral amino acid transporter [L-type amino acid transporter (LAT)] is a transmembrane-type protein which engages in cellular uptake of neutral amino acids such as glycine, alanine, leucine, isoleucine, valine, serine, threonine, cysteine, asparagine, glutamine, methionine, phenylalanine, tyrosine, tryptophan, histidine, L-DOPA, and the like. The present inventors have been searching various LATs and have found LAT1 and isoforms thereof, namely, LAT2, LAT3, and LAT4.
Among them, LAT1 is a 12-pass transmembrane spanning domain protein that has the ability to transport large neutral amino acids such as leucine, isoleucine, valine, methionine, phenylalanine, tyrosine, tryptophan, histidine, and the like; and L-DOPA, Na+ coexisting with an amino-acid transport activating factor, 4F2hc independently. Moreover, LAT1 is mainly expressed in vivo such as in placenta, spleen, colon, testis, and a blood-brain barrier. Also, the expression of LAT1 is recognized in a human signet ring cell carcinoma cell line, a small cell lung cancer cell line, a melanoma cell line, a neuroblastoma cell line, and further confirmed by immunohistochemical staining in tumor tissues of kidney cancers, bladder cancers and prostate cancers (refer to Patent Literature 1, Non-Patent Literature 1 and Non-Patent Literature 2). The suppression of the expression of LAT1 by suppressors such as melphalan, BCH, and the like remarkably reduces the growth rate of cultured cells that are expressing LAT1. Therefore, the intracellular uptake of essential amino acids by LAT1 is absolutely imperative for cell proliferation, and it has been thought that the expression of LAT1 at a high level helps cancer cells to obtain a superior nutrition state compared to normal cells, so cancer cells are predominant in cell proliferation over normal cells.
LAT2, which is the isoform of LAT, has a transmembrane structure that spans the intracellular membrane 12 times as well as LAT1, and has a substrate selectivity in a broad range that allows a Na+ independent transport of all the neutral amino acids, including small amino acids such as glycine, alanine, serine, threonine, cysteine, asparagine, and glutamine in addition to large amino acids, by coexisting with 4F2hc. It has been confirmed that LAT2 is expressed in normal tissues in the brain, skeletal muscles, kidney, jejunum, ileum, testis, and placenta, but not in tumor tissues (refer to Patent Literature 2 and Non-Patent Literature 3). Since the affinity of LAT2 with a substrate is low compared to that of LAT1, LAT2 is thought to play a role in transporting neutral amino acids in cells of normal tissues that do not require active cell proliferation.
Furthermore, the present inventors have found various transporters including a sodium independent small neutral amino acid transporter that transports L- and D-forms amino acids (refer to Patent Literature 3), and also a transporter of which substrates are cystine, basic amino acids, and neutral amino acids (refer to Patent Literature 4). A transporter is defined as a transmembrane-type protein having a function of taking up various substances that are essential to cells in a substance-specific way, so it is essential for the maintenance and proliferation of body tissues. Therefore, many methods for the diagnosis of diseases by utilizing various transporters have been proposed. For example, methods including one to detect the presence or absence of the expression of a protein or to determine the amount of the protein in samples derived from organs or the part of thereof containing tumor cells, tumor tissues, or tumors (refer to Patent Literature 1); a method for the diagnosis of lung/thoracic diseases characterized by using the antibody of an anion transporter (refer to Patent Literature 5); a method to diagnose whether patients are affected in integration disorder syndrome by a quantitative value of a protein, a fragment thereof, or nucleic acid of an erythrocyte urea transporter as an index (refer to Patent Literature 6); a method for discriminating benignancy from malignant tumor overexpressed GLUT-1 by preparing a cytological preparation from body cavity fluid and comparing the expression level of GLUT-1 to a tissue sample obtained from the section of non-malignant tissues of the same type of tissues using an antibody that can bind to the transmembrane glucose transporter GLUT-1 (refer to Patent Literature 7) have been reported.
Mainly three biomarkers intended for use in the treatment of breast cancer are known, that is, estrogen receptor (ER), progesterone receptor (PgR) and HER2 (a receptor of growth factor, which is also referred to as EGFR2). In case an immunohistochemistry or FISH method is carried out for each biomarker in tumor tissues and finds positive cases, the treatment method targeted to a corresponding biomarker has been chosen for cases showing positive to each biomarker. On the other hand, a breast cancer that shows negative for all of these markers (three proteins, these are an estrogen hormone receptor, progesterone hormone receptor and HER2 are not expressed in tumor cells in cancer) is referred to as triple negative breast cancer, which accounts for 12 to 25% of the total population of breast cancer cases (refer to Non-Patent Literature 7 to 10). Appropriate treatment method for the triple negative breast cancer has not been developed still now. The patients have poor prognosis compared to those to breast cancer that show positive to either of three markers (refer to Non-Patent Literature 9 and 10).
Also, a histopathology method using specimens stained with conventional HE (hematoxylin-eosin) is carried out for the discrimination between malignant ductal carcinoma in situ (hereinafter sometimes referred to as DOTS) and benign intraductal breast papilloma (intraductal breast papilloma) in intraductal breast tumors. However, well-defined discrimination elements are not clear, so there are many lesions that are difficult to discriminate between benignancy and malignancy. Therefore, the development of a marker that can provide more explicit discrimination is needed.